The present invention relates to a method of detecting an antigenic substance by immunodiffusion using as a reagent the antigenic substance to be detected labelled by a chromogenic enzyme and also to a detection material which is directly used for the detection of antigenic substance. More specifically, the present invention relates to a method of detecting antigenic substances which comprises addition of a test sample containing an antigenic substance to be detected and a material containing the antigenic substance to be detected labelled by a chromogenic enzyme to a supporting material uniformly containing therein the antibody or antiserum against the antigenic substance to be detected, an immunoreaction (or antigen-antibody reaction) to form an antigen-antibody complex, color reaction by the chromogenic enzyme in the complex, and the measurement of the degree of the diffusion of the enzyme in the complex.
Furthermore, the invention also relates to a material for detecting antigenic substances comprising a supporting material uniformly containing therein the antibody or antiserum of the antigenic substance to be detected and a material containing the antigenic substance to be detected labelled by a chromogenic enzyme.
The terms "antigenic substance" and "antigen" used in this invention mean not only a so-called complete antigen which possesses immunogenic reactivity but also haptens which do not possess immunogenicity but possess antigenic reactivity.
It therefore includes various antigens, for example serum protein antigens such as .alpha.-fetoprotein, .gamma.-globulin G, .gamma.-globulin E, etc.; protein antigens such as an enzyme antigen, a toxin antigen, etc.; lipid antigens such as glycocalyx (cell wall polysaccharide), dextran and levan; other polysaccharide antigens comprising saccharides, a phospholipid, a lipopolysaccharide, a neutral lipid, etc; hapten antigens such as an azoprotein, a sulfurized substance, a 2,4-dinitrophenol protein, a steroid protein, a penicillin protein complex, a synthetic polypeptide, etc.; antinucleic acid antibody-producing nucleic acid antigens such as ribosomal, nuclear and phage antigens as well as, DNA, RNA, synthetic polynucleotides, a protein conjugate of a component constituting nucleic acid, etc; and blood group antigens such as an ABO antigen, a Lewis antigen, a forseman antigen, an MN antigen, a Rh antigen, etc.
The qualitative or quantitative detection of various antigenic substances is not only useful for the prophylaxis and diagnosis of various diseases and physiological abnormalities related to these substances but also widely utilized in some other research field for example, the determination of the molecular weight of proteins. For example, if the quantitative detection of a very small amount of human .alpha.-fetoprotein is possible, it is useful for the diagnosis of hepatoma and the quantitative detection of a very small amount of human .gamma.-globulin E or human gamma globulin E is likewise useful for the diagnosis of the diseases caused by immunological disorders.
Hitherto, as a method of detecting antigenic substances, for example, methods utilizing an antigen-antibody precipitation reaction or a hemagglutination or a complement fixation reaction are known. The method utilizing a hemagglutination or a complement fixation reaction is an indirect method and is such methods, the operation is complicated and the quantitative treatment thereof frequently encounters difficulty.
As the method utilizing the precipitation reaction, there is known an immunodiffusion method wherein the degree of the diffusion of the antigen-antibody complex formed by a precipitation reaction such as an antigen-antibody reaction, etc., is measured without separating the antigen-antibody complex and a method wherein the antigen-antibody complex formed by a precipitation reaction is separated by means of centrifugal precipitation, adsorption, gel filtration, etc., and thereafter the content of the unreacted antigen or the formed antigen-antibody complex is measured. The aforesaid immunodiffusion methods are classified according to the manner of diffusion, for example, into a single diffusion, a double radial diffusion and an immunoelectrophoresis (so-called rocket electrophoresis). Further, there is an immunodiffusion method employing staining and an immunodiffusion method using a radioisotope-labelled compound. These immunodiffusion methods are simple in operation but are usually inadequate for a quantitative measurement of a very small amount of an antigen for their limited detection sensitivity.
Furthermore, even in the immunodiffusion method employing an additional staining method, the quantitatively detectable range is at most about 10 .mu.g./ml. Also, the immunodiffusion method using a radioisotope-labelled compound is disadvantageous in the operation process for its time consumption with obtaining the result and for the necessity of specific reagents and apparatus due to the radioactive substance. On the other hand, as the aforesaid latter method wherein the antigen-antibody complex formed is separated, there are known various methods such as a method using a radioisotope-labelled antigen (radioimmunoassay), a method using an enzyme-labelled antigen (enzyme immunoassay), etc. Among these methods, the radioimmunoassay has now been frequently used since the method can detect quantitatively a very small amount of an antigen. However, these methods are complicated in operation since it is inevitable in these methods to separate the antigen-antibody complex formed by a precipitation reaction from the unreacted antigen. Furthermore, since in the radioimmunoassay, it is necessary to use specific reagents and apparatus due to the use of radioactive substances, this method has the faults that the operation is complicated, it takes a long period of time to obtain the result and the cost is very high.